Systematic Characterization of SLC2A9 (Glut9) Variants Associated with Serum Uric Acid Levels

Abstract

Glut9 is a voltage sensitive urate transporter, mainly expressed in the kidney, the liver and the intestines. It constitutes a major determinant of serum uric acid levels and variants have been associated with hyperuricemia and hypouricemia. In particular, several Glut9 loss-of-function mutations were identified as responsible for familial hypouricemia type II in humans and hyperuricemia in dogs. Moreover, several single nucleotide polymorphisms have been associated with lower serum urate. To get insights in Glut9 transport characteristics, we analyzed the expression and function of each Glut9 variant. GLUT9 variants were prepared by site-directed mutagenesis and expressed in Xenopus laevis oocytes. Fourty-eight hours later the function of each individual variant was studied using (i) 14C-urate uptake (n=30, 3 batches) in presence of 400µM urate and (ii) two-electrodes voltage clamp in presence of 400µM and 1mM urate (n=15, 3 batches). Surface expression was assessed by immunostaining and biotinylation (n=3). We found decreased urate transport by flux studies for most of the variants. We could further differentiate two different behaviors among the variants: those harboring poor surface expression, and, more interestingly, those keeping full cell surface expression, but harboring decreased function. We further studied the latter by electrophysiology and observed decreased inward currents measured in presence of 400 µM urate, an observation that could be partially reversed in presence of 1mM urate. These results show that most of loss-of-function Glut9 variants are non-functional due to poor surface expression. We identified three variants involved in urate binding. Systematic analysis of loss-function Glut9 mutants brings insight in Glut9 structure – function.